Journal: JCI Insight
Article Title: Deletion of SH2D5 alleviates epileptic seizures and NMDAR expression via autophagic degradation of STAT1
doi: 10.1172/jci.insight.191347
Figure Lengend Snippet: ( A ) Quantification of STAT1 mRNA levels via qPCR. ( B and C ) Representative Western blot images ( B ) and quantification ( C ) of the protein level of STAT1 at different timepoints after CHX treatment. ( D ) Western blot images showing the STAT1 protein levels in the CQ, MG132, and DMSO treatment groups. ( E and F ) Representative Western blot images ( E ) and quantification ( F ) of the protein level of Beclin1. ( G ) Representative immunofluorescence images showing SH2D5 and LC3 colocalization. Scale bar: 100 μm. Arrowheads: the cell that expresses LC3. ( H and I ) Representative Western blot images ( H ) and quantification ( I ) of LC3-II/LC3-I levels. ( J and K ) Representative Western blot images ( J ) and quantification ( K ) of STAT1 levels after RAPA administration. ( L and M ) Representative Western blot images ( L ) and quantification ( M ) of GluN1, GluN2A, and GluN2B levels after RAPA administration. The data are presented as mean ± SEM. Unpaired t test in A , F , I , K , and M ; 2-way ANOVA with Tukey’s multiple-comparison test in C . *** P < 0.001.
Article Snippet: Primary antibodies against the following proteins were used for Western blotting: anti-SH2D5 rabbit antibody (1:1,000; PA5-101883, Thermo Fisher Scientific), anti-STAT1 rabbit antibody (1:2,000; 10144-2-AP, Proteintech), anti-STAT3 rabbit antibody (1:1,000; 10253-2-AP, Proteintech), anti–p-STAT3-705 rabbit antibody (1:1,000; EPR23968-52, Abcam), anti–p-STAT3-727 rabbit antibody(1:1,000; E121-31, Abcam), anti-GAPDH rabbit antibody (1:4,000; 10494-1-AP, Proteintech), anti-Calnexin rabbit antibody (1:2,000; 10427-2-AP, Proteintech), anti-GluA1 rabbit antibody(1:2,000; 25012-1-AP, Proteintech), anti-GluA2 rabbit antibody(1:2,000; 11994-1-AP, Proteintech), anti-GluN1 rabbit antibody (1:2,000; 27676-1-AP, Proteintech), anti-GluN2A rabbit antibody (1:2,000; 28571-1-AP, Proteintech), anti-GluN2B rabbit antibody (1:1,000; 21920-1-AP, Proteintech), anti-Beclin1 rabbit antibody (1:2,000; sc-48341, Santa Cruz Biotechnology), and anti-LC3 rabbit antibody (1:2,000; 18725-1-AP, Proteintech).
Techniques: Western Blot, Immunofluorescence, Comparison
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